Paradoxical enhancement of interleukin-2-mediated cytotoxicity against K562 cells by addition of a low dose of methotrexate

Summary In vitro effects of methotrexate (MTX) on interleukin-2(IL-2)-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) were studied. PBMC were incubated with human recombinant IL-2 (25 U/ml) for 72 h; during the last 24 h, various concentrations (10 pM–1 µM) of MTX were added to the culture. Cytotoxicity against k562 cells was measured by a 4-h⁵¹Cr-release assay. The IL-2-mediated cytotoxicity was paradoxically increased at around a concentration (10 nM) MTX. Such a low concentration of MTX showed no anti-proliferative effect on cell growth. This enhancement with 10 nM MTX was shown only in an E-rosette⁺ (E⁺) population, but not in E-rosette^– (E^–). In addition, when E⁺ cells were treated with an anti-CD16 monoclonal antibody plus complement after incubation with IL-2 and MTX, MTX-induced enhancement was lost, suggesting that an E⁺CD16⁺ cell population was mainly involved in this augmentation. Positively sorted E⁺CD16⁺ cells showed similar enhancement of cytotoxicity after treatment with IL-2 plus MTX. On the other hand, MTX treatment did not show the phenotypical changes including of the E⁺CD16⁺ cells, indicating that this treatment did not affect the differentiation and proliferation of the specific cell subset. Our results indicate that a low dose of MTX could have a role in the regulation of immunological anti-cancer surveillance systems through the natural killer and lymphokine-activated cytotoxic cells.This work was supported in part by a Grant-in-Aid for Cancer Research (1–10) from the Ministry of Health and Welfare in Japan     

Production of hydroxyl radicals and their disassociation from myocardial cell injury during calcium paradox.

The production of hydroxyl radicals during calcium paradox injury was investigated by measuring the production of 2,5-dihydroxybenzoic acid (2,5-DHBA) from salicylate. Four groups of rats were analyzed. In the first group, isolated hearts were perfused with calcium-free medium for 10 minutes followed by perfusion with medium containing Ca++ for 10 minutes. In the other groups, 0.25 microM N,N'-diphenyl-1,3-phenylenediamine (DPPD), 80 microM cytochrome c, or 450 U/ml catalase was added. Coronary effluent was analyzed for the presence of 2,5-DHBA, and tissue sections were examined using light microscopy. In the first group, 2,5-DHBA production began during the calcium-free period, peaked tenfold 60-90 sec. into the Ca repletion period, and declined thereafter. The increase in 2,5-DHBA was accompanied by severe cell damage. Cytochrome c reduced 2,5-DHBA production, and catalase almost completely inhibited 2,5-DHBA production, while DPPD had no effect on 2,5-DHBA production. None of the three additives provided any complete morphological protection. The data provide evidence for the production of hydroxyl radicals during calcium-paradox injury, that their production is dependent upon the presence of hydrogen peroxide, and that cell damage in the calcium paradox is not primarily mediated by the extracellular hydroxyl radicals.     

Distribution of the pulmonary artery and vein of the orangutan lung

The distribution of the pulmonary artery and vein of the orangutan lung was examined. The right pulmonary artery runs obliquely across the ventral side of the right bronchus at the caudally to the right upper lobe bronchiole. It then runs across the dorsal side of the right middle lobe bronchiole. Thereafter it runs obliquely across the dorsal side of the right bronchus, and then along the dorso-medial side of the right bronchus. This course is different from that in other mammals. During its course, it gives off branches which run mainly along the dorsal or lateral side of each bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole, then along the dorso-lateral side of the left bronchus, giving off branches which run along each bronchiole. The pulmonary veins run mainly the ventral or medial side of, along or between the bronchioles. In the left lung, the left middle lobe vein has two trunks; one enters the left atrium, and the other enters the left lower lobe pulmonary venous trunk. This is also different from that found in most mammals. Finally, the pulmonary veins enter the left atrium with four large veins.     

Cryofixed,freeze-dried and paraffin-embedded skin enables successful immunohistochemical staining of skin basement membrane antigens

Summary Conventional chemical fixation and paraffin-embedding procedures give good preservation of morphology, although the antigenicity of many proteins in the tissue sample is destroyed. On the other hand, fresh frozen sections can preserve the antigenicity, but provide poor morphological preservation. To overcome this dilemma, cryofixation and freeze drying were used on human skin tissue, applying methodology which has only been used to study lymphoid tissue. First, fresh human skin was cryofixed in liquid isopentane (–160° C) cooled by liquid nitrogen. The skin was then freeze-dried at –40° C and 10^–2 atmospheric pressure for 72 h, followed by embedding in paraffin. Sections 4 m thick taken from this cryofixed, freeze-dried, and paraffin-embedded skin were stained with hematoxylin-eosin or used for immunolabeling with antibodies against basement membrane antigen, including type IV and type VII collagen, bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and GB3 antigen. The morphological preservation of these sections was as good as that of routine formalin-fixed and paraffin-embedded skin sections. The basement membrane was clearly immunostained with all antibodies used, and the intensity of the reaction was as strong as that seen in frozen sections. Evaluation of antigen distribution in conjunction with the detailed skin structure was therefore possible in the same sections.A part of this work was presented at the 90th Annual Meeting of the Japanese Dermatological Association, Kyoto, Japan, April, 1991     

Degradation of Dehydrodivanillin by Anaerobic Bacteria from Cow Rumen Fluid

Dehydrodivanillin (DDV; 0.15 g/liter) was biodegradable at 37°C under strictly anaerobic conditions by microflora from cow rumen fluid to the extent of 25% within 2 days in a yeast extract medium. The anaerobes were acclimated on DDV for 2 weeks, leading to DDV-degrading microflora with rates of degradation eight times higher than those initially. Dehydrodivanillic acid and vanillic acid were detected in an ethylacetate extract of a DDV-enriched culture broth by thin-layer, gas, and high-performance liquid chromatographies and by mass spectrometry.     

Inheritance of hydronephrosis in the inbred mouse strain DDD

The mode of inheritance of hydronephrosis was investigated by crossing inbred DDD mice having a high incidence of hydronephrosis and C57BL/6 mice having normal kidneys. In the males, incidences of hydronephrosis in F1 animals were intermediate between the two parental strains at a rate of 32.6% in (DDD x C57BL/6)F1 and 23.4% in reciprocal F1. The same tendency was observed in F2 male animals. In BCF1 males, the number of affected mice was higher in (C57BL/6 x DDD) F1 x DDD (72.4%) than in (DDD x C57BL/6)F1 x C57BL/6 (11.1%). A few affected mice were found among the females of hybrids F1, F2 and BCF1. These results suggested that hydronephrosis in the DDD strain of mice was controlled by polygenes, and that male hormones may have some effect on the occurrence of hydronephrosis.     

Regulatory Mechanisms of Reproductive Effort in Plants II. Plasticity in Reproductive Energy Allocation and Propagule Output of Glycine max Merr. (Leguminosae) Cultivated at Varying Densities and Nitrogen Levels

Abstract Plasticity in growth, reproductive energy allocation (RA), and reproductive output were studied in Glycine max Merr. Cv. Enrei (Leguminosae) grown under varying densities and soil nitrogen levels.
Marked plastic responses were detected in individual biomass, the patterns of resource allocation to total reproductive structures (RA) and also to propagules, reproductive outputs, and propagule weight under changing densities and soil nitrogen levels. Plants cultivated at higher densities exhibited proportionately lower individual biomass, lower RA, lower seed output, and smaller seed size in response to increasing density and decreasing soil nitrogen levels, although some deviations were observed, especially in the highest density plot with no fertilization. Differences due to different N-levels were not as great as those to changing density, which may in part be due to the fact that soybean has nitrogen-fixing bacteria in root tubercles, just as in any other Leguminosae. Fecundity was also maintained at the similar high rates of 80–97% in all plots examined, although slight but steady decreases were noted with increasing density. This resemblance in fecundity may be due to its strong inbreeding system.
Another important finding was that seed production under limited resource availability, notably lack of ample solar radiation due to strong interference at higher density plots, is exceedingly costly. This was most clearly exhibited by a sharp increase in relative energy partitioning to a single propagule in response to the increased density, the relative energy cost to a single propagule (RA) increasing from one to seven-fold. The results obtained in this study coincide well with the findings made in other plants, e.g., Helianthus annuus, Oryza sativa , and Coix ma-yuen , with the same experimental designs.     

Properties of three creatine kinases MM from porcine skeletal muscle

The properties of three fractions (FI, FII, and FIII) of porcine creatine kinase MM, which have been isolated by isoelectric focusing, were compared. Sugars were not detected in them. Their carboxyl-terminal sequences were identical and were determined to be -Thr-Lys by digestion with carboxypeptidases A and B. Immunodiffusion and competitive radioimmunoassay could not differentiate the three fractions from one another. Their amino-terminal sequences revealed that they had different primary structures. At residue 1, although all the three fractions had Pro, FI and FIII had an additional amino acid, Ser. At residue 23, only FI had Leu in addition to Ser, the amino acid common to the three fractions. These results indicate that differences among the three fractions of porcine creatine kinase MM are based on differences in the primary structures of the subunits in their dimer structures, and confirm the conclusion that FII is a homodimer and FI and FIII are heterodimers, which was reported in the preceding paper [Takasawa, T. & Shiokawa, H. (1983) J. Biochem. 93, 383-388].